JPS60243100A - Novel protein and controller for plant virus containing it - Google Patents

Abstract

NEW MATERIAL:A basic protein contained in tissue of a plant belonging to the genus Mirabilis. Color reaction: ninhydrin reaction positive, phenol-sulfuric acid reaction negative, isoelectric point: pI=9-10. Molecular weight: 2.42X10<4> (by SDS-polyacrylamide gel electrophoresis). USE:A controller for plant virus. Effective especially against insect in fectious viruses, showing systemically its antiviral activity, having no problems with respect to safety. PREPARATION:Tissue of roots, stalks, leaves, or others of a plant belonging to the genus Mirabilis is extracted with an aqueous solvent, the extract is passed through a column, its active ingredient is adsorbed, so that it is purified.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a basic protein obtained from the tissue of a plant belonging to the genus Oscillaria and having an anti-plant virus effect, and a plant virus disease control agent containing this as an active ingredient. Regarding. These rules can be widely used in the field of pickling or solidification for the purpose of controlling viral diseases.

(Prior art) Tobacco, tomatoes, green peppers, cucumbers, watermelons, radish, Chinese cabbage, etc. grown in fields, rice paddies, or various facilities are infected with tobacco mosaic virus (hereinafter referred to as TM'v).
Mosaic disease, wilt disease caused by viruses such as cucumber mosaic virus C (hereinafter referred to as Head), cucumber green spot mosaic virus C (hereinafter referred to as CGMMV), potato Y virus (hereinafter referred to as 1'VY), and turnip mosaic virus (hereinafter referred to as Tu MY), They often suffer from diseases such as Ezo disease and suffer significant damage. These pathogenic viruses are transmitted to other crops,
It exists in weeds, tree seedlings, soil, etc., and is transmitted through contact during management work, sweat absorption by insects, etc. Conventional measures to control these viral diseases in crops include removing or reducing the source of the virus, using barrier crops or coverings to prevent the introduction of virus-carrying insects, cultivating methods such as cultivating resistant crops, and using soil. Indirect control techniques have primarily been used, such as killing virus vectors with disinfectants or insecticides. Direct control agents for plant viruses include sodium alginate agent (Patent No. 717594, Ministry of Agriculture, Forestry and Fisheries registration No. 13440) and shiitake mycelium culture extract (Patent No. 1012014, Ministry of Agriculture, Forestry and Fisheries registration No. 15).
No. 584), but all of these drugs target only contact-transmitted TMV and do not have the ability to penetrate the plant body, making them ineffective against insect-transmitted viruses that cause significant damage to many crops. be.

(Problems to be Solved by the Invention) An object of the present invention is to provide a chemical substance that does not have these drawbacks of conventional plant virus disease control agents and is particularly effective against insect-transmitted viruses.

(Means for solving the problems) The present inventors have developed a drug that has fewer problems in terms of safety, exhibits antiviral activity systemically, and is effective against insect-transmitted viruses. For the purpose of development, we screened a large number of substances, mainly natural products, and found that the roots, stems, nine leaves, and soils of plants belonging to the genus Mirabilis in the Nyctaginaceae family of the order Chenopodiales. It was discovered that a protein contained in the tissue exhibits significant antiviral activity.

Plants belonging to the genus O. jalap include Mirabilis jalap, Mirabilis longiflora, and U. longiflora.
ora.

There are several types of plants, such as M. mirabilis, M. nyctaginea, and M. nyctaginea. The crude extract or diluted purified product obtained by the method is used for tobacco, tomato, green pepper, cucumber, watermelon, radish,
When sprayed or applied to the leaves of Chinese cabbage, etc., or absorbed from underground, 'rMv, Ch(V,
It can effectively prevent the onset of infections such as C (WIJV, PVY, TuMV, etc.).In particular, the antiviral activity of plant components of the genus P.
Unlike proteins, it is expressed systemically in treated plants, so it is also highly effective in preventing virus transmission by sweat-absorbing insects such as aphids. The present inventor has experimentally confirmed these facts and has completed the present invention.

(Example) Examples relating to the extraction and purification of the protein of the present invention mainly using the root of Oscilos as a material and the physical and chemical properties of the purified product will be described in detail.

Air-dried or freeze-dried product of roots of O. mirabilis I Kv
(approximately 5 ~ fresh weight equivalent) was ground in a pulverizer to form a powder, and then 0.5% of 2-mercaptoethanol was added. OI
201 ml of M phosphate buffer (pH 7,2) was added, and the mixture was ground using a mixer or homogenizer. The obtained homogenate was centrifuged for 5,000×gjlS minutes and separated into a supernatant and a precipitate.

An additional lot of the above extraction medium was added to the precipitate, stirred well, and centrifuged at 5,000×g for 15 minutes. The supernatants obtained from both centrifugation treatments were combined, and ammonium sulfate (hereinafter referred to as ammonium sulfate) was added to make it 50% saturated.
The supernatant portion was collected by centrifugation at ,000xg for 15 minutes. Ammonium sulfate was further added to the supernatant to achieve 90% saturation, and centrifugation was performed in the same manner to obtain a precipitate. This precipitate was dissolved in a small amount of deionized water, dialyzed against deionized water, and then lyophilized. The crude extract is hereinafter referred to as 90% ammonium sulfate precipitation. Approximately 7.5 f of 90% ammonium sulfate precipitate was obtained per Ikg of dry root of Ossifolia.

A crude extract having antiviral activity can also be obtained by precipitation with acetone, ethanol, or the like.

That is, acetone or ethanol is added to the centrifuged supernatant of the above buffer homogenate of Osprey roots at a concentration of 50% or 75% (volume percentage), the resulting precipitate is collected by centrifugation, and then freeze-dried for crude extraction. (hereinafter referred to as 50% acetone precipitation or 75% ethanol precipitation). Dried root of Oshirobana 11
50% acetone precipitation per g4 38.4r, 75%
Ethanol precipitation is 53.8? Obtained. In addition to these precipitation methods, it is also possible to obtain a crude extract by adding the centrifuged supernatant of the crude juice to a cation exchange carrier such as CM-Sepharose described below to adsorb the active ingredient, and then eluting and concentrating as appropriate. can.

Column chromatography was performed on the above precipitate with ammonium sulfate, acetone, ethanol, etc. to purify the active ingredient. First, extract the crude extract at 0.01M5pH6,o(7)! j
After dissolving in phosphoric acid and fur, CM-Sepharose CL-
6B (trade name, Pharmacia) column to adsorb the active ingredient, and eluted with a NaCl solution with a linear concentration gradient of 0.15 to 0.4M.
Activity was observed in the 18 MNaCl elution fraction. The active fraction was collected and dialyzed against 0.01M, pH 7.0 phosphate buffer, then passed through a DEAR-Sepharose (trade name, Pharmacia) column equilibrated with the same buffer, and flowed out from the column. Both fractions were collected, and after adjusting the pH to 6.0, the above CM-Sepharose column chromatography was performed again. This column chromatography yielded a single peak showing antiviral activity.
This was collected, dialyzed against deionized water, and then freeze-dried to obtain a purified product. This purified product showed a typical protein absorption spectrum with a ratio peak of 87.5 cm per dry root IKf of Oscillaceum, and the color reaction was positive by the ninhydrin method and negative by the phenol-sulfuric acid method. Amino acid analysis of the hydrochloric acid hydrolyzate of this substance revealed that it is a lysine-rich polypeptide, as shown in Table 1. Furthermore, when the isoelectric point of this substance was investigated using carrier ampholine (a trade name of LKB Company), it was found that the pI
Values of =9 to lO were obtained. The molecular weight of this substance is 8D8-
2.42X according to polyacrylamide gel electrophoresis
This value was in good agreement with the molecular weight of 2.41×104 calculated from Table 1. Furthermore, according to ultracentrifugation analysis, the sedimentation coefficient was 52oW=2.5. From the above results, this substance can be said to be a basic protein.

Table-1 Arginine 6 Aspartic acid 22 Threonine 24 Selin 19 Glutamic acid 19 Print 8 Green screen 12 Alanine 18 Ballin 12 Cysteine l Methionine 3 Inleucine 16 0 Ishin 18 Tyrosine lO Phenylalanine lO tryptophan l Note 1) Tryptophan was analyzed by absorbance method.

This article describes a method for controlling viral diseases using active ingredients obtained from propagated plants of the genus Porphyra.

When applying this active ingredient to crops, it does not necessarily have to be a purified product; it may be a centrifuged supernatant of a ground juice of plant tissue,
Ammonium sulfate precipitation, acetone precipitation.

An unpurified product such as ethanol precipitate may be appropriately diluted and used. These can be used as aqueous solutions, wettable powders, etc. by adding a spreading agent, a surfactant, a filler, a stabilizer, etc. according to a general method for producing agricultural chemicals. TMV,
When targeting contact-transmissible viruses such as CGMMV, it is effective to spray prior to any management work that involves direct contact with the crops, such as moving, soiling, budding, fA pulling, and harvesting in nurseries or fields. CMV, PVY, T
When targeting insect-borne viruses such as uMv,
Effective control can be achieved by treating crops at intervals of approximately one week, prior to the arrival of vector insects. No phytotoxicity was observed in the plants treated with the main rule, and there were no negative effects on the growth of the plants.

(Effects of the Invention) The effects of the invention will be described below with reference to an experimental example of a plant virus control test conducted using the active ingredient of P. mirabilis.

Harmful experiment example 1 2-mercapto x ta/
Antiviral activity of the centrifuged supernatant of the homogenate (referred to as buffer extract) obtained by adding O,OXM phosphate buffer containing 0.1% of -1k, the above-mentioned ammonium sulfate precipitate, acetone precipitate or ethanol precipitate, and the purified product. was assayed for TMV, CGMMV, CMV and TuMV. For the assay, plants that produce localized spots by inoculation with the virus, i.e., tobacco (variety Xanthi NC) for TMV, CGMMV
against Datura, CMV and TuM
For V, tobacco (variety Prite Yellow) was used.

These plants were grown in pots with a diameter of 12 crn, and the test solution was applied with a paintbrush to fresh leaves bordering the main vein on the front or back side of the expanded leaves, and water was applied to one side of the leaves as a control. One day after sample treatment, each virus was inoculated onto the entire upper surface of the leaves.

The inoculated virus concentration is TMV 0.05 μ9/ml.
, CMV is 5μf/ml, CGMM'v and TuM'v
The sap of infected leaves was diluted 1,000 times and 100 times, respectively. Three to seven days after virus inoculation, the number of spots appearing on the inoculated leaves was counted, and the control value was calculated using the following formula.

The test results are shown in Table-2. Various samples of P. elegans showed extremely high control value for all combinations of viruses and plants tested.

(J's 1 Chemical 9) Experimental Example 2 An example will be described showing that when a plant is treated with an extract of a plant belonging to the genus Porphyra, the effects of the active ingredients contained therein are systematically expressed in the plant. 57 days after sowing, 5 lower leaves of tobacco (Xanthi n.c.) with a plant height of about 456 nm were treated with 50% acetone precipitate of P.
Spray at a concentration of 0.05μf/ml on the top three leaves one or three days after spraying.
was inoculated by smear. Water was sprayed as an untreated control. TM
Three days after V inoculation, the spots that appeared on the inoculated leaves were counted, and the control value was calculated according to Example 1. As is clear from Table 3, when some leaves of tobacco are treated with this active ingredient, the number of spots is reduced even on untreated leaves of the same individual.
It can be concluded that the effect of this active ingredient is systemic.

Table 3 Classification Days after spraying Number of spots/leaf control value Untreated control plot -521,5- 50% acetone precipitation sprayed plot (invention) 1 128.8
75 Experimental Example 3 The preventive effect of the present active ingredient against virus transmission to aphids was tested as follows. , 90% on the entire stem of potted tobacco (Burley 21) and turnip (Kanamachi Kokabu).
Ammonium sulfate precipitate or 50% acetone precipitate was sprayed at a concentration of 1/ml, and one day after the spraying, K CMV and pvy Aruiha TuM'v were inoculated using green peach aphids.

Water was sprayed on the untreated control plot. Aphid inoculation is 2
Wingless insects of the green peach aphid fed for 2 hours at 5°C were infected with diseased plants (CM'v o and PVY grouper A:l,
TuMV was applied to turnips (turnips) and allowed to absorb sap for 10 minutes, and then released to test plants at a rate of 5 heads/plant. The presence or absence of disease onset was observed for 2-L after inoculation with the virus, and the effectiveness of the treatment was determined from a comparison of the incidence of disease between treated and untreated plots. As shown in Table 4, the active ingredients are CMV and PVY.

It showed a high preventive effect against any aphid infection of TuMV.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption of the purified product of the present invention, vector (concentration: i, s s s++ y/ml o, o IM !j phosphoric acid, 77-+ pH 6,0) at 6°.

Claims (1)

[Scope of Claims] 1. The following a, which is obtained by extracting the tissue of a plant belonging to the genus Oscillina with an aqueous solvent and purifying the extract.
A basic protein identified by the physical and chemical properties of e. a. The ultraviolet absorption spectrum has a peak at a wavelength of 280 nm. b. Ninhydrin reaction is positive and phenol-sulfuric acid reaction is negative. C0 isoelectric point pI = 9-1O d, molecular weight is 2.42×10 as determined by 5DS-polyacrylamide gel electrophoresis. e. The sedimentation coefficient determined by ultracentrifugation analysis is S = 2.5. 2. A plant virus disease control agent characterized by containing as an active ingredient a basic protein identified by the following physical and chemical properties extracted from the tissue of a plant belonging to the genus Porphyra. Also, the ultraviolet absorption spectrum has a wavelength of 280 nmlC peak. b. Ninhydrin reaction is positive and phenol-sulfuric acid reaction is negative. C1 isoelectric point pI = 9 to lOd, molecular weight is 2.42XlO' as measured by 5DS-polyacrylamide gel electrophoresis. e, The sedimentation coefficient by ultracentrifugation analysis is S2Ow = 2.5
It is.